This grant application seeks funding for the development of cell-type-specific retroviral vectors for the uture in vivo gene therapy of HIM infection. In the past seven years, my laboratory has developed retroviral vectors, derived from spleen necrosis virus, SNV, which display the antigen binding site of an antibody (single chain antibodies, scAs) on the viral surface. We have shown that such particles enable an efficient, cell-type-specific gene transfer into human cells. In this application, experiments are proposed to further develop this gene transfer system using targeting envelope useful for the transfer of therapeutic genes into human cells of the hematopoietic system. The following targeting ligands will be displayed on the final surface: (i) an anti-CD34 scA to target human hematopoietic stem cells; (ii) an anti-HIV-SU scA to specifically transduce genes into HIV-1 infected cells; (iii) the complete surface unit peptide of the HIV-1 envelope. SNV particles displaying this peptide would have the same host range like HIV-1. Experiments will be performed to increase the gene transfer efficiency of this cell-type-specific gene delivery system and to test cell-type specificity in tissue culture experiments. Furthermore, experiments will be performed in SCID mice to test the efficiency and cell-type specificity in vivo. Wild-type SNV infects a large variety of species and appears to utilize a house-keeping receptor for virus entry like other retroviruses investigated. Although SNV is not infectious on human cells, efficient infection of targeting ligand displaying vectors into human cells is dependent on the co-presence of a fully functional SNV wildtype envelope protein. To understand the mechanism of virus entry of targeting vectors, experiments will be performed to first clone and analyze the receptor gene of cells susceptible to SNV infection (e.g., dog D17 cells) followed by the cloning and characterization of the homologous receptor gene from human cells, which appears to be mutated preventing efficient binding of wild-type SNV. Insight into the mechanism of the entry of targeting vectors will certainly help to make further vector improvements. The development of this cell-type-specific gene transfer system will make it possible to in vivo transduce and analyze the effect of many therapeutic genes developed by the scientific community for gene therapy of HIV-1 infections. RESUME: The proposal seeks funds to develop retroviral vectors displaying single chain antibodies (scAs), which will direct the infection of specific target cells expressing the antigen. A specific goal is to develop vectors displaying scA against the human CD34 antigen to be able to deliver HIV-1 resistant genes into the hemopoietic stem cells. The rationale being that the benefits from currently available viral protease and RT inhibitors are short lived due to the emergence of drug resistant mutants, and the fact that high virus load persists in clinically latent AIDS. This laudable goal to develop alternative therapeutics using "gene antivirals" however is not without major challenges.